JPH0259667A - Method of inspecting of syphilis antibody utilizing enzyme antibody method - Google Patents

Abstract

PURPOSE:To specifically detect the presence or absence of a syphilis infection by fixing a Treponema Pallidum antigen onto a solid phase and detecting the syphilis antibody of IgA or IgG 1 to 4 in serum by an enzyme antibody method. CONSTITUTION:The Treponema Pallidum antigen subjected to a heating treatment and ultrasonic wave treatment is fixed to a solid phase such as microplate. Serum albumin, etc. are brought into reaction after washing and again the washing is executed. The diluted serum to be inspected is added to the antibody to effect reaction. Further, the diluted antibody of the anti-human IgG 1 to 4 or IgA is added thereto after washing to effect reaction. An enzyme labeling antibody is added thereto after washing to effect reaction. A substrate soln. corresponding to enzyme is added to enzyme to effect reaction and color development; thereafter, the degree of color development is measured. The presence or absence of the in-blood antibody to the Treponema Pallidum is specifically detected with the high sensitivity.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to the use of Treponema pallidum (
Immunoglobulin (Ig) that constitutes the antibody against TP)
This article relates to a syphilis antibody testing method that uses an enzyme-antibody method to detect syphilis.

[Prior Art] Conventionally, as with other infectious diseases, measurement of antibodies in the blood has occupied a major position as a method of confirming syphilis infection, in addition to judgment based on clinical symptoms. Particularly in the case of syphilis, in recent years there has been a tendency for so-called syphilis syphilis, which is not accompanied by clinical symptoms, to increase, and its diagnosis is largely dependent on the latter. A large number of methods have been put into practical use to determine syphilis infection. Historically, there has been a method using cardiovirin, a type of phospholipid, but the Wallaselman reaction, which is famous as a typical test method, has become less common in recent years due to the complexity of the reaction system, and has been replaced by an alternative method. Glass plate method <VDRL method), RPR method, etc. are used. Although these two methods have a long history, they have not lost their usefulness even today because of their simplicity, rapidity, and excellent reproducibility of results. However, on the other hand, antibody detection sensitivity is low,
It has the disadvantage that it cannot necessarily be said to be a specific response to syphilis, as it has cross-reactivity with some diseases including systemic lupus erythematosus (SLE). In order to eliminate these drawbacks, Treponema, the pathogen of syphilis,
Several methods have been devised to use pallidum (TP) as an antigen.

That is, the PTA-ABS method, which applies a fluorescent antibody method, and the TPHA method, which utilizes indirect hemagglutination reaction, etc.

[Problems to be Solved by the Invention] Although the PTA-ABS method is effective for measuring various classes of antibodies described later, it has some difficulty in reproducibility of the results, and it is difficult for the person in charge of testing to make judgments. Its use tends to decrease in recent years due to its drawbacks such as the tendency for individual differences to occur.
be. On the other hand, the TPHA method can be used to determine the presence or absence of syphilis infection.
This is a very simple and effective method, and is currently widely used worldwide, along with the glass plate method and RPR method mentioned above. However, with the spread of the T PHA method, several problems have arisen.

The most important problem is that the TPHA method has high sensitivity for TKG antibodies among immunoglobulins, but low sensitivity for IgM antibodies, which are antibodies produced in the early stages of infection.

At present, serological testing methods for syphilis require the use of two or more surgical methods with different essences or mechanisms.
According to the regulation, STS using cardiopyrin as an antigen
Many laboratories are implementing a combination of the treponemal antigen method and the method using treponemal antigens.
The difference in sensitivity of the HA method to antibodies causes two clinical complications. First, there are cases in which the TPHA method is unable to capture IgM antibodies that occur in the early stages of syphilis infection, and only the glass plate method gives a positive result, and the discrepancy between the two methods causes confusion in clinical diagnosis.

Second, there is the reality that IgG antibodies hardly decrease even with treatment, so the TPHHA method continues to show positive results even after syphilis patients have received adequate treatment and clinical symptoms have disappeared. .

Recently, the TPH,A method has been improved, and some similar methods are commercially available, but although the sensitivity to IgM antibodies has increased, a decrease in antibody titer due to fP during treatment cannot be observed even with these methods. The percentage of people who do so is small.

Each of the above testing methods is a so-called screening method for determining the presence or absence of syphilis infection.

By the way, the purpose of measuring antibodies in blood is not only for this screening method but also to determine the therapeutic effect by examining temporal changes in antibodies in blood, as partially described above. Regarding this point, it has been reported for about 10 years that IgM antibodies among the antibodies produced by syphilis infection can serve as an indicator, and the glass plate method, RPR method, and IgM antibody have been reported.
The PTA-ABS method and the like aimed at gM antibody detection meet that purpose. However, as mentioned above, these methods cannot always be completely trusted due to reasons such as disease specificity, sample detection sensitivity, reproducibility of results, and complexity of procedures. Then fractionated TPHA
A method has been devised to detect IgM antibodies and IgM antibodies in serum.
Because it requires a method to separate G antibodies in advance, the method is complicated and special equipment must be installed to perform it as a routine work in a laboratory. It is only carried out at a testing center. Moreover, since it is impossible to measure in a state close to so-called real time, its application range is mainly limited to research purposes.

In order to overcome this drawback, a method for measuring IgM antibodies among immunoglobulins (Ig) using an enzyme antibody method has been developed. Since this method is a very new method, its evaluation has not been completely determined, but as described later (see bottom of page 19), IgM antibodies show trends parallel to treatment. .

A decrease in TgM antibodies due to treatment is not observed in all cases. In particular, it has been observed that there are many cases in which gM antibodies do not decrease in late-stage cases in which there are no symptoms and the period of infection is unclear. On the other hand, IgG antibodies have been investigated mainly using the TPHA method, but they have been treated as antibodies that hardly decrease even with treatment and do not show negative effects even after thorough anti-inflammatory therapy. .

Serum actually collected over time from patients after treatment was separated into an IgM fraction and an IgG fraction by means such as gel filtration, and the antibody titers in both fractions were investigated. The TPHA antibody titer in the fraction decreases, but that in the I2c fraction does not change. However, one of the methods for measuring syphilis antibodies, T.
When measured using the PIA method (immunoadhesion reaction),
It was observed that the antibody titer of the IgG fraction was decreased as well as the antibody titer of the IgM fraction. This suggests that there is a decrease in one or more of the subclasses rgG1 to rgG4 constituting IgG due to treatment.

[Means for Solving the Problems] The present invention was developed to solve the above problems, and
The syphilis antibody testing method using the enzyme-antibody method of the present invention involves adsorbing antibodies against the bacterial cells of the syphilis pathogen Treponema pallidum or their crushed components or human immunoglobulin onto a solid phase such as a microplate. Each syphilis antibody of immunoglobulin IgA or IgG1 to 4 is detected by implantation or chemical bonding and using an enzyme antibody method.

Methodologically, the present invention uses an enzyme antibody method, but in principle there are cases in which the antigen Treponema pallidum is bound to a solid phase, and cases in which antibodies of each of the above classes are bound to a solid phase. Furthermore, in both cases, there are various methods, such as directly binding the enzyme to the antigen or antibody, or mediated by biotin or avidin.

1 Effect] As described above, the present invention is directed to adsorbing, embedding, or chemically adsorbing the bacterial cells of Treponema pallidum, the pathogen of syphilis, or their fragments, or human immunoglobulin antibodies onto a solid phase such as a microplate. immunocropurin IgA or IgG1-4 using enzyme antibody method.
Since each syphilis 1g antibody is detected, the syphilis antibody in the test serum can be tested based on the degree of color development.

[Example] Examples of the invention are listed below.

In addition, during the following operations, the medium used for cleaning is 0.0
Phosphate buffer saline, (PBS-tween) containing 5% concentration of Tween 20, and various proteins such as serum albumin, for dilution of reagents such as antibodies, serum, and Treponema pallidum antigens. PBS-tween containing various animal serums or skim milk powder is used.

In addition, enzymes for labeling include peroxidase or
Alkaline phosphatase or the like is used, and a phenylenediamine solution or aminosalicylic acid containing hydrogen peroxide as a substrate is used.

Example A.

1. Immobilize Treponema pallidum antigen at an optimal concentration on a solid phase such as a plate. Trebonema, which has been subjected to a certain heat treatment and ultrasonic treatment, is used as the antigen, and is reacted at an alkaline pH for a certain period of time, and then adsorbed onto a solid phase.

2. After washing with medium (PBS-Tween), bring the solution containing proteins such as serum albumin to an alkaline pH.
The plate is incubated at a constant temperature for a certain period of time to fill in the parts of the plate that are not covered with treponemal antigens. (This operation makes it possible to suppress non-specific reactions in the reaction system.) 3. After washing with medium, add the sample diluted to the optimum concentration and allow to react at a constant temperature for a certain period of time.

4 Wash with medium, add anti-human IgG1-4 or IgA antibodies (polyclonal or monoclonal antibodies) diluted to the optimal concentration with the solution used in step 3, and react at a constant temperature for a certain period of time, 5. After washing with medium again, add the enzyme-labeled antibody to the optimal concentration using the solution used in step 3 (in a system using polyclonal antibodies, use the antibody corresponding to 1 g of animal derived from the antibody,
In a system using monoclonal antibodies, antibodies against mouse Ig are reacted at a constant temperature for a certain period of time.

6. Wash the plate with a medium, add the substrate solution corresponding to the enzyme used in 5, react at a constant temperature for a certain period of time to develop color, then add a reaction stop solution and measure the degree of color development.

In addition, in the operation step 4 of Example A, anti-human I
If enzyme-labeled antiserum treatment such as anti-human I [Gl-4, IgA, etc.] is carried out instead of each antibody (monoclonal or polyclonal antibody) such as gG1-4, anti-human IgA, it can be used without using a secondary antibody. Proceed to the step of adding substrate.

Example B.

1. Anti-human IgG1 to 4 subclass antibodies at optimal concentrations;
Alternatively, anti-human IgA antibodies are allowed to adhere to the plate under alkaline pit.

2. After washing with a medium, a solution containing a protein such as animal serum albumin is allowed to react under alkaline PI at a constant temperature for a certain period of time to cover the exposed portion of the plate that is not covered with antibodies.

3. Wash with a medium, add the test serum diluted to the optimum concentration with a medium containing proteins such as animal serum albumin, and react at a constant temperature for a certain period of time.

4. After washing with medium, add Trebonema antigen solution at optimal temperature and react at constant temperature for a certain period of time.

5. After washing with a medium, react with an enzyme-labeled anti-treponemal antibody (including antibodies in the form of so-called F(ab)2 with the Fc portion removed) at a constant temperature for a certain period of time.

6. After washing with medium, add a substrate solution corresponding to the enzyme, react at a constant temperature for a certain period of time, and measure the degree of color development.

Example C1 1-3; Same as B; 4; After washing with medium, add enzyme-labeled Trebomane antigen solution at optimal concentration and react at a certain temperature for a certain period of time; 5; After washing with medium, correspond to enzyme Add the prepared substrate solution, react at a constant temperature for a certain period of time, and measure the degree of color development.

Examples.

1. Immobilize Treponema pallidum antigen (TI antigen) at an optimal concentration on a solid phase such as a plate.

2. Add diluted test serum to the TP antigen on the same phase and react at a constant temperature for a certain period of time.

3. After washing thoroughly with medium, add an optimal concentration of the antibody (anti-human IgG1-4 or IgA anti-human IgG1-4 or IgA anti-human IgG labeled with biotin).
React at a constant temperature for a certain period of time. (However, the secondary antibody at this stage can be used as it is, or it can be treated with an enzyme to remove the FC region and converted into F(ab)2 form.) 4. Wash this well with medium. After that, a certain amount of an avidin-labeled enzyme such as peroxidase or alkaline phosphatase is reacted at a certain temperature for a certain period of time.

5. After washing thoroughly with medium, add the substrate solution and measure the degree of color development. The method of immobilization on the solid phase is to perform any one of the following operations (1) to (2), followed by operation (2).

■ Adsorb the optimal concentration of TP antigen onto the solid phase under neutral or alkaline pFI (adsorption) ■ Drop the TP antigen suspended in an organic solvent such as chloroform onto the solid phase and air dry it (embedding) ■ Dissolve a crosslinking agent such as glutaraldehyde or carbodiimide in an organic solvent or buffer such as chloroform or water, add it to the solid phase, air dry, and react with the TP antigen under neutral or alkaline pH (binding).
Synthetic polymers such as polylysine, natural proteins such as bovine serum albumin, low-molecular compounds such as various amino acids, and substances with functional groups that can react with cross-linking agents are adsorbed onto the solid phase, or by methods such as ■. After the above operations, the remaining exposed solid phase is filled with serum albumin, immunoglobulin, etc. under alkaline pH to initiate the reaction. increase the specificity of

As shown in the drawing, the enzyme antibody method used in this example uses a secondary antibody (an antibody against human immunoglobulin) bound to biotin and an enzyme bound to avidin, so-called biotin/avidin. system is used.

Methods using this biotin-avidin system are generally considered to have excellent sensitivity and specificity, but as far as syphilis antibody detection is concerned, the non-specific reaction is strong and it is not suitable for practical use. .

However, as a result of various improvements, it became possible to minimize non-specific reactions, making it fully practical.

In other words, biotin, which is used in conventional tests and research,
The non-specific reaction that appeared strongly with the avidin method.

This can be reduced by the following two points.

■ Proteins such as serum albumin fill the in-phase surface that is not covered with TP antigens; ■ In addition to biological components such as proteins or sugars, lower the ionic strength and pH of the buffer containing surfactants. do.

Example E.

1 to 3 are the same as Example B.

4. After washing with medium, add biotinylated Trebonema antigen solution and react at a constant temperature for a certain period of time.

5. After washing with medium, add an avidin-treated enzyme solution and react at a constant temperature for a certain period of time.

6. Same as Example B.

Example F.

1 to 4 are the same as Example B.

5. After washing with medium, add anti-Trebonema antibody solution F (a
b') f) (including those formed in the form) are added and allowed to react at a constant temperature for a certain period of time.

6. After washing with medium, enzyme-conjugated rabbit Ig
Add antiserum against the target and allow to react at a constant temperature for a certain period of time.

7. After washing with medium, react with a substrate solution corresponding to the enzyme used, and measure the degree of color development.

Example G.

1 to 4 are the same as Example B.

5. After washing with medium, add a solution of biotinylated anti-Trebonema antibody F(ab') f) and allow to react at a constant temperature for a certain period of time.

6. After washing with medium, add an avidin-treated enzyme solution and react at a constant temperature for a certain period of time.

7. Add a substrate solution corresponding to the enzyme used to react, and measure the degree of color development.

[Effects of the Invention] Clinically determining syphilis depends on the clinical symptoms, but in addition to this, serological diagnosis, that is, the presence or absence of antibodies against Treponema pallidum in the blood, is performed. However, the majority of patients currently targeted for treatment are cases without clinical symptoms, and therefore, unlike the most common infectious diseases, serological diagnosis accounts for a very large proportion; If the diagnosis is positive, a diagnosis of syphilis will be made immediately. Furthermore, patients diagnosed with syphilis naturally receive treatment, and the means of determining the effectiveness of treatment at this time are to measure the disappearance of clinical symptoms and the degree of decrease in the amount of antibodies in the blood. In other words, if a decrease in the amount of antibodies in the blood is not observed even after the clinical symptoms have subsided, it is difficult to diagnose that the patient is completely cured, and it is difficult to know when to discontinue treatment. Therefore, measuring the amount of antibodies in the blood plays a very important role in determining the effectiveness of syphilis treatment.

At present, it can be said that measuring antibodies in the blood is essential as a marker for determining therapeutic efficacy. The present invention is related to this point, and has the following effects: l) The presence or absence of syphilis infection can be measured with higher specificity and sensitivity.

2) 1gG subclass or Ig after treatment of syphilis patients
Tracking changes in blood levels of A antibodies provides information on therapeutic efficacy. Furthermore, at this time,
By tracking changes in IgA antibodies in addition to each IgG subclass, it is possible to follow up on treatment effects for more than 90% of patients.

By the way, disease diagnosis methods using immunological techniques largely depend on the specificity of antigen-antibody reactions. However, antibodies against substances that have multiple antigenic sites, such as bacteria, react not only with related species but also with other species, and the latter In this case, it becomes necessary to carry out an operation to separate them, which makes measurement and interpretation of the obtained results more complicated. Therefore, it is desirable to have a measurement method with low cross-reactivity with other diseases, in other words, with high specificity. Therefore, it is very important to examine whether the method of the present invention reliably provides a response that satisfies specificity for syphilis. Diseases that are subject to this problem include, in particular, systemic lupus erythematosus (SLE) and rheumatoid arthritis. When these diseases were studied, the following findings were found.

That is, in SLE patients, the STS method using cardiolibin as an antigen showed a fairly positive reaction, but the 1 gM antibody measurement method and the method of the present invention all showed negative results. However, rheumatoid arthritis patient serum sometimes showed non-specific reactions using the IgM antibody assay, and occurred at a higher rate than the non-specific reactions observed using the tg^ antibody assay. On the other hand, in the 1gG subclass antibody assay, no such non-specific reaction occurred, demonstrating that the antibody has excellent disease specificity.

Water droplets have another advantage over IgM antibody assays.

That is, if a patient with syphilis is treated, the blood 1
In 90% of cases, the gM antibody level decreases, and in the remaining 10% there is no change, making the therapeutic effect unclear. However, with water droplets, antibody reduction was observed in 9.20% of patients, which is more improved than when IgM antibodies are used as a marker (however, when used in combination with the IBM antibody measurement method, it is 9.5%, which is higher). information).

[Brief explanation of the drawing]

The drawing is an explanatory diagram of a method for testing syphilis antibodies using a biotin-conjugated antibody and an avidin-conjugated enzyme in Example C of the present invention.

Claims (1)

[Claims] Antibodies against the bacterial cells of Treponema pallidum, the pathogen of syphilis, or their constituent components, or human immunoglobulin (Ig) are adsorbed, embedded, or chemically bonded to a solid phase such as a microplate, and then enzyme-linked antibodies are used. A syphilis antibody testing method using an enzyme antibody method to detect immunoglobulin IgA or IgG1-4 syphilis antibodies.